ï› The sequence GGG was added at the 5' and 3' ends of primers to enhance restriction enzymes to digest exactly at their proper cut sites and avoid flanking.

2.1.3. Culture Media:

2.1.3.1. LB Liquid medium:

To 900 ml of distilled water, add:

Bacto-tryptone (Difco) 10.0 g

Bacto-yeast extract (Difco) 5.0 g

NaCl (Sigma) 10.0 g

Distilled water 900.0 L

The pH was adjusted to 7.0 with 5N NaOH, then the volume of the solution was completed to 1 liter, and the medium was sterilized by autoclaving for 20 minutes at 121&deg;C. For preparing solid medium, the same recipe of 2xYT-medium was prepared, followed by the addition of bacto-agar (Difco) up to 1.5% (w/v), sterilized by autoclaving for 20 minutes at 121&deg;C then allowed to cool to 50&deg;C before pouring into petridishes. After autoclaving, filtered sterilized ampicillin (100 mg/ml) or Kanamycin (50mg/ml) was added to a final concentration (100 ï­g/ml)or (50ï­g/ml) respectively.

2.1.3.2. Ampicillin (Sigma) (100 mg/ml):

Ampicillin 1.0 g

Distilled water 10.0 ml

The solution was sterilized by filtration through a 0.2ï­m filter, then divided into 0.5 ml aliquots and stored at -20&deg;C.

2.1.3.3 Kanamycin (sigma) ( 50mg/ml)

Kanamycin 0.5g

Distilled water 10.0ml

The solution was sterilized by filtration through a 0.2ï­m filter, then divided into 0.5 ml aliquots and stored at -20&deg;C.

2.1.9. Reagents and Solutions (Sambrook et al., 1989):

The pH was adjusted to 7.0, then the volume of the solution was completed to 100 ml, and sterilized by filtration through a Nalgene filter (0.45µm pore size). The acrylamide was stored in a dark bottle at room temperature.

ï› 10% Ammonium persulfate:

Ammonium persulfate 0.1 g

Distilled water to 1.0 ml

The solution was divided to aliquots and stored at 4&deg;C for up to several weeks.

ï› Chloroform-iso-amyl alcohol (Chisam):

Chloroform 24.0 volumes

Iso-amyl alcohol 1.0 volume

ï› Coomassie destaining solution:

Methanol 45.0 ml

Distilled water 45.0 ml

Glacial acetic acid 10.0 ml

ï› Coomassie staining solution (SDS-PAGE staining solution):

Coomassie Brilliant Blue R250 0.25 g

Methanol 45.00 ml

Distilled water 45.00 ml

Glacial acetic acid 10.00 ml

The solution was mixed well and filtered through a Whatman No. 1 filter and stored in an aluminum foil wrapped bottle at room temperature.

ï› Ethylene diamine tetra acetic acid (EDTA), 0.5M, pH 8.0:

EDTA 18.61 g

Distilled water 80.00 ml

The pH was adjusted to 8.0 with 10N NaOH, the volume was completed to 100ml, dispensed into aliquots and sterilized by autoclaving.

ï› Ethidium bromide, 10 mg/ml:

Ethidium bromide 0.1 g

Distilled water 10.0 ml

The solution was dissolved by stirring on magnetic stirrer for several hours, and stored at room temperature in an aluminum foil wrapped tube.

ï› Isopropylthio-ï¢-D-galactoside (IPTG), (M.W = 238.8):

IPTG 1.0 g

Distilled water to 5.0 ml

The solution was sterilized by filtration through a 0.2 ï­m filter, dispensed into 1ml aliquots and stored at -20&deg;C.

ï› Phenol: (equilibrated to pH &gt;7.8):

The phenol was melted at 68&deg;C then hydroxyquinoline was added as an antioxidant to a final concentration of 0.1 %. Equal volume of 0.5 MTris.Cl pH 8.0 was added to the liquified phenol and stirred for 15 minutes at room temperature. After the two phases had separated, as much as possible of the aqueous (upper) phase was aspirated off. Equal volume of 0.1M Tris.Cl pH 8.0 was added to the phenol, stirred, the phases were left to separate, and the upper aqueous phase was aspirated off as before. This step was repeated for several times until pH of phenolic phase was &gt;7.8. The final aqueous phase was discarded and 0.1M Tris.Cl pH 8.0 was added. The phenol was then stored in this form in a light-tight bottle at 4&deg;C for periods of up to 1 month.

ï› Phenol-Chloroform:

Equilibrated phenol 1 volume

Chloroform 1 volume

Stored under 0.01M Tris.Cl pH 7.6 at 4&deg;C in dark glass bottle.

ï› Potassium acetate buffer (5M):

Potassium acetate 49.1g

Distilled water 80.0 ml

ï› Ribonuclease, pancreatic (RNase A), 10 mg/ml:

RNase A 10 mg

Distilled water 1 ml

The solution was boiled for 15 minutes, then cooled to room temperature before dispensing into 100 μl aliquots and stored at -20&deg;C.

ï› 3M Sodium acetate, pH 5.2:

Sodium acetate 20.4 g

Distilled water 40.0 ml

The pH was adjusted to 5.2 with glacial acetic acid then the volume was completed to 50 ml with distilled water and dispensed into aliquots and sterilized by autoclaving.

ï› 10% Sodium dodecyl (lauryl) sulfate (SDS):

SDS 5 g

Distilled water 40 ml

The solution was heated to 68&deg;C, pH was adjusted to 7.2 by adding a few drops of concentrated HCl and the volume was adjusted to 50 ml with distilled water.

ï› 10N Sodium hydroxide:

Sodium hydroxide 20 g

Distilled water to 50 ml

ï› Solution I: (For alkaline lysis of plasmid DNA mini preparation):

glucose 0.9 g (50mM final)

1MTris-HCl, pH 8.0 2.5 ml (25 mM Final)

0.5M EDTA, pH 8.0 2.0 ml (10mM final)

Distilled water to 100.0 ml.

The solution was autoclaved for 20 minutes at121&deg;C, and stored at 4&deg;C.

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